Metoprine, a histamine N-methyltransferase inhibitor, attenuates methamphetamine-induced hyperlocomotion via activation of histaminergic neurotransmission in mice.

Metoprine increases the content of histamine in brain by inhibiting histamine N-methyltransferase (HMT), a centrally acting histamine degrading enzyme. We present data demonstrating that pretreatment with metoprine attenuates the hyperlocomotive effects of METH in mice using a multi-configuration behavior apparatus designed to monitor four behavioral outcomes [horizontal locomotion, appetitive behavior (food access), and food and water intake]. Metoprine pretreatment itself induced hyperlocomotion in mice challenged with saline during the large part of light phase. The trend was also observed during the following dark phase. This is the first report that metoprine has a long-lasting locomotor stimulating property. Similarly, in a tail suspension test, a single injection of metoprine significantly reduced total time of immobility in mice, consistent with the idea that metoprine possesses motor stimulating properties. Metoprine pretreatment did not affect other aspects of behavior. Metoprine did not affect the appetitive and drinking behavior while exerted an effect on stereotypy. No stereotyped behavior was observed in mice pretreated with vehicle followed by METH, while stereotyped sniffing was observed in mice pretreated with metoprine followed by METH. The metoprine pretreatment attenuated METH-induced hyperlocomotion during the first 2 h of light phase, suggesting that metoprine-induced locomotor stimulating property might be different from that of METH. The hypothalamic content of histamine (but not its brain metabolite) was increased after metoprine or METH administration. Both METH and metoprine reduced dopamine and histamine turnover in the striatum and the nucleus accumbens and the hypothalamus, respectively, and there is a significant metoprine pretreatment x METH challenge interaction in the histamine turnover. It is likely that metoprine may attenuate METH-induced hyperlocomotion via activation of histaminergic neurotransmission. Metoprine also might induce a long-lasting locomotor stimulating effect via a putative mechanism different from that whereby METH induces the locomotor stimulating effect.

6-Hydrophobic aromatic substituent pyrimethamine analogues as potential antimalarials for pyrimethamine-resistant Plasmodium falciparum.

The series of des-Cl (unsubstituted) and m-Cl phenyl analogues of PYR with various flexible 6-substituents were synthesized and studied for the binding affinities with highly resistant quadruple mutant (QM) DHFR. The derivatives carrying 4 atoms linker with a terminal carboxyl substituted on the aromatic ring exhibited good inhibition to the QM enzyme and also showed effective antimalarial activities against resistant P. falciparum bearing the mutant enzymes with relatively low cytotoxicity to mammalian cells. The X-ray crystallographic analysis of the enzyme-inhibitor complexes suggested that the hydrophobic substituent at 6-position was accommodated well in the hydrophobic pocket and the optimal length of the flexible linker could effectively promote the binding of the terminal carboxyl group to the key amino acid residues, Arg59 and Arg122.

A novel prediction approach for antimalarial activities of Trimethoprim, Pyrimethamine, and Cycloguanil analogues using extremely randomized trees.

Malaria is still one of the most serious diseases in tropical regions. This is due in part to the high resistance against available drugs for the inhibition of parasites, Plasmodium, the cause of the disease. New potent compounds with high clinical utility are urgently needed. In this work, we created a novel model using a regression tree to study structure-activity relationships and predict the inhibition constant, Ki of three different antimalarial analogues (Trimethoprim, Pyrimethamine, and Cycloguanil) based on their molecular descriptors. To the best of our knowledge, this work is the first attempt to study the structure-activity relationships of all three analogues combined. The most relevant descriptors and appropriate parameters of the regression tree are harvested using extremely randomized trees. These descriptors are water accessible surface area, Log of the aqueous solubility, total hydrophobic van der Waals surface area, and molecular refractivity. Out of all possible combinations of these selected parameters and descriptors, the tree with the strongest coefficient of determination is selected to be our prediction model. Predicted Ki values from the proposed model show a strong coefficient of determination, R2=0.996, to experimental Ki values. From the structure of the regression tree, compounds with high accessible surface area of all hydrophobic atoms (ASA_H) and low aqueous solubility of inhibitors (Log S) generally possess low Ki values. Our prediction model can also be utilized as a screening test for new antimalarial drug compounds which may reduce the time and expenses for new drug development. New compounds with high predicted Ki should be excluded from further drug development. It is also our inference that a threshold of ASA_H greater than 575.80 and Log S less than or equal to -4.36 is a sufficient condition for a new compound to possess a low Ki.

New derivatives of the antimalarial drug Pyrimethamine in the control of melanoma tumor growth: an in vitro and in vivo study.

BACKGROUND: The antimalarial drug Pyrimethamine has been suggested to exert an antitumor activity by inducing apoptotic cell death in cancer cells, including metastatic melanoma cells. However, the dose of Pyrimethamine to be considered as an anticancer agent appears to be significantly higher than the maximum dose used as an antiprotozoal drug. METHODS: Hence, a series of Pyrimethamine analogs has been synthesized and screened for their apoptosis induction in two cultured metastatic melanoma cell lines. One of these analogs, the Methylbenzoprim, was further analyzed to evaluate cell-cycle and the mechanisms of cell death. The effects of Methylbenzoprim were also analyzed in a severe combined immunodeficiency (SCID)-mouse xenotransplantation model. RESULTS: Low dose of Methylbenzoprim was capable of inducing cytotoxic activity and a potent growth-inhibitory effect by arresting cell cycle in S-phase in melanoma cells. Methylbenzoprim was also detected as powerful antineoplastic agents in SCID-mouse although used at very low dose and as a single agent. CONCLUSIONS: Our screening approach led to the identification of a "low cost" newly synthesized drug (methylbenzoprim), which is able to act as an antineoplastic agent in vitro and in vivo, inhibiting melanoma tumor growth at very low concentrations.

Metoprine induced behavioral modifications and brain regional histamine increase in WAG/Rij and Wistar rats.

The effects of metoprine, an inhibitor of histamine N-methyltransferase, on open field activity and brain regional histamine (HA) content were examined in rats with mixed, absence and audiogenic, epilepsy (WAG/Rij-AGS), rats with audiogenic epilepsy (Wistar-AGS) and in non-epileptic control rats (Wistar-nAGS). HA content was increased by metoprine (20mg/kg, i.p.) in the cortex, striatum, thalamus, hypothalamus and hippocampus of the rats from all three tested groups. However, WAG/Rij rats showed a lower rate of metoprine-induced HA accumulation in the striatum and thalamus than Wistar rats. For the open field test, the main effect of metoprine (20mg/kg, i.p.) was a general increase of locomotor activity although distinctive features, such as hyperlocomotion and exaggerated sniffing, were characteristic for the epileptic rats (WAG/Rij-AGS and Wistar-AGS, respectively). Individual rats from all the groups showed stereotyped behavior of shuttle type and head bobbing. Electroencephalographic data obtained in WAG/Rij-AGS rats confirmed that metoprine-induced behavioral activation was accompanied by suppression of spike-wave discharges, the main hallmark of absence seizures. Taken together, these results show that inhibition of the histamine catabolism may induce motor activation of particular patterns in epileptic rats and provoke stereotyped behavior.

Virtual screening identification of nonfolate compounds, including a CNS drug, as antiparasitic agents inhibiting pteridine reductase.

Folate analogue inhibitors of Leishmania major pteridine reductase (PTR1) are potential antiparasitic drug candidates for combined therapy with dihydrofolate reductase (DHFR) inhibitors. To identify new molecules with specificity for PTR1, we carried out a virtual screening of the Available Chemicals Directory (ACD) database to select compounds that could interact with L. major PTR1 but not with human DHFR. Through two rounds of drug discovery, we successfully identified eighteen drug-like molecules with low micromolar affinities and high in vitro specificity profiles. Their efficacy against Leishmania species was studied in cultured cells of the promastigote stage, using the compounds both alone and in combination with 1 (pyrimethamine; 5-(4-chlorophenyl)-6-ethylpyrimidine-2,4-diamine). Six compounds showed efficacy only in combination. In toxicity tests against human fibroblasts, several compounds showed low toxicity. One compound, 5c (riluzole; 6-(trifluoromethoxy)-1,3-benzothiazol-2-ylamine), a known drug approved for CNS pathologies, was active in combination and is suitable for early preclinical evaluation of its potential for label extension as a PTR1 inhibitor and antiparasitic drug candidate.

The synthesis and antifertility evaluation of 3 analogs of pyrimethamine on male rat reproductive indices.

Side effect assessment of medicaments on fertility indices may be used as a guide in the development of male contraceptive agents. In this study, 3 analogs of pyrimethamine were synthesized and evaluated for antifertility activity on reproductive indices of male rats. Test compounds were administered in a dosage of 50 mg/kg every other day till 60 days. On the 50th day, the fertility of rats was tested. On the 60th day, the gonadosomatic index and the serum testosterone content were determined. Iso-butyloxy and tertiary-butyloxy caused 40% and 11% reduction in sperm viability, respectively. They also significantly reduced fertility indices. Consequently, iso-butyloxy can be one of the best nominees in this class of compound and a suitable candidate for assessment of mechanism involved in future research activity. To synthesize a more effective agent, increasing the lipophilicity may play a major role in the development of more potent promising male contraceptive agents.

Particular interaction between pyrimethamine derivatives and quadruple mutant type dihydrofolate reductase of Plasmodium falciparum: CoMFA and quantum chemical calculations studies.

Comparative molecular field analysis (CoMFA) was performed on twenty-three pyrimethamine (pyr) derivatives active against quadruple mutant type (Asn51Ile, Cys59Arg, Ser108Asn, Ile164Leu) dihydrofolate reductase of Plasmodium falcipaarum (PfDHFR). The represented CoMFA models were evaluated based on the various three different probe atoms, C(sp3) (+1), O(sp3) (-1) and H (+1), resulting in the best model with combined three types of probe atoms. The statistical results were r(2)(cv) = 0.702, S(press) = 0.608, r(2)(nv) = 0.980, s = 0.156, and r(2)(test-set) = 0.698 which can explain steric contribution of about 50%. In addition, an understanding of particular interaction energy between inhibitor and surrounding residues in the binding pocket was performed by using MP2/6-31G(d,p) quantum chemical calculations. The obtained results clearly demonstrate that Asn108 is the cause of pyr resistance with the highest repulsive interaction energy. Therefore, CoMFA and particular interaction energy analyses can be useful for identifying the structural features of potent pyr derivatives active against quadruple mutant type PfDHFR.

Histaminergic involvement in neuropathic pain produced by partial ligation of the sciatic nerve in rats.

BACKGROUND AND OBJECTIVES: Because the histaminergic system in the brain is involved in regulation of pain, the relationship between central histaminergic activity and neuropathic pain is of interest. METHODS: Neuropathic pain was induced in rats by partial ligation of the left sciatic nerve, and changes in the extracellular concentration of histamine in the right striatum were examined by a microdialysis procedure 2 weeks later. The nociceptive threshold was determined with von Frey tests, and effects of histaminergic ligands were examined. RESULTS: Although the extracellular concentration of histamine did not differ between the sham-operated and ligated groups, histaminergic activity assessed by metoprine-induced accumulation of histamine was facilitated in ligated animals. The metoprine treatment ameliorated neuropathic pain in ligated animals, although the agent did not affect the threshold in sham-operated rats. Either intracerebroventricular (ICV) administration of histamine (30 microg) or intraperitoneal (IP) administration of L-histidine (370 mg/kg) decreased the nociceptive threshold in ligated rats. However, a high dose of histamine (180 microg ICV) increased the nociceptive threshold. Ranitidine (100 microg ICV), an H2 antagonist, increased the threshold, whereas pyrilamine (15 microg ICV), an H1 antagonist, showed no remarkable change. Administration of thioperamide (30 microg ICV), an H3 antagonist, increased the threshold, although systemic administration of the agent (3.6 mg/kg IP) decreased it. CONCLUSIONS: Blockade of supraspinal histamine H2 receptors or stimulation of spinal H3 receptors may contribute to alleviation of neuropathic pain.

Histaminergic modulation of acoustically induced running behavior in rats.

Explosive running is a reliable initial component of audiogenic seizures (AS) induced by acoustic stimulation in genetically prone rodents. This profound locomotor activation is usually considered as a convulsive manifestation of AS although some studies attribute running to a panic-like response. Increase in central histamine activity has been shown to suppress clonic and tonic seizures. The present study examined the involvement of histaminergic mechanisms in the expression of running component of AS. Metoprine, an inhibitor of histamine-N-methyltransferase, was used to increase brain histamine level. Running was induced 4 and 24 h after intraperitoneal injection of metoprine or vehicle in rats of different strains. A brief sound stimulation elicited running followed by clonic-tonic convulsions in Krushinsky-Molodkina (KM) rats or running alone in AS-prone Wistar and WAG/Rij rats. In KM rats, metoprine exerted opposite effects on the main phases of AS. It increased the duration of running and decreased the duration and severity of clonic-tonic convulsions. In Wistar rats, metoprine produced a remarkable aggravation of running leading to its 2- to 3-fold prolongation. In WAG/Rij rats with mixed seizures (absence and audiogenic), the drug caused either aggravation or suppression of running behavior. These results suggest specific role for histaminergic system in the expression of behavioral components of AS. Suppressive role of histamine in clonic-tonic seizures is associated with facilitation of running suggesting specific effects of histamine on brainstem neuronal networks underlying these phases of AS. Possible roles of histaminergic mechanisms in seizure, motor and aversive aspects of sound-induced running are discussed.

[New chemical constituents from Radix Polygoni Multiflori after processing].

OBJECTIVE: To study the new chemical constituents from Radix Polygoni Multiflori after processing. METHODS: Various kinds of chromatographic methods were used to deparate the chemical constituents from Radix Polygoni Multiflori after processing. Their structures were determined by NMR and MS spectral data. RESULTS: The two new compounds were 2,3-dihydro-3,5-dihydroxy-6-meth-yl-4 (H)-pyran-4-one(I) and 5-hydruoxymethyl-furfuran(II). CONCLUSION: It is the first time that compound I and 1I were isolated from Polygoni.

Evaluation of the activities of pyrimethamine analogs against Plasmodium vivax and Plasmodium falciparum dihydrofolate reductase-thymidylate synthase using in vitro enzyme inhibition and bacterial complementation assays.

Pyrimethamine analogs were examined as potential agents against vivax malaria using a bacterial surrogate system carrying Plasmodium vivax dihydrofolate reductase-thymidylate synthase (PvDHFR-TS), in which the PvDHFR complemented chemically knocked out host dihydrofolate reductase. The system was initially tested with P. falciparum dihydrofolate reductase-thymidylate synthase and was found to have good correlation with the parasite-based system. The 50% inhibitory concentrations derived from PvDHFR-TS-dependent bacteria were correlated with their corresponding inhibition constants (Ki) from an enzyme inhibition assay, pointing to the likelihood that the potent enzyme inhibitors will also have potent antimalarial activities. Active compounds against both wild-type and S58R S117N (SP21) double-mutant P. vivax include analogs with structures which can avert a steric clash with the asparagine (S117N) side chain of the mutant, similar to those found for homologous Plasmodium falciparum mutants, raising the possibility that the same compounds can be developed against both types of antifolate-resistant malaria. This rapid and convenient drug screening system should be useful for development of new antifolates against P. vivax, for which a continuous culture system is not yet available.

Mutant Gly482 and Thr482 ABCG2 mediate high-level resistance to lipophilic antifolates.

Cellular uptake of hydrophilic antifolates proceeds via the reduced folate carrier whereas lipophilic antifolates enter cells by diffusion. Recently we have shown that transfectant cells overexpressing the mutant G482 ABCG2 displayed 120-6,250-fold resistance to hydrophilic antifolates than untransfected cells upon 4 h drug exposure, but lost almost all their antifolate resistance upon 72 h drug exposure (Shafran et al. in Cancer Res 65:8414-8422, 2005). Here we explored the ability of the wild type (WT) R482-as well as the mutant G482-and T482 ABCG2 to confer resistance to lipophilic antifolate inhibitors of dihydrofolate reductase (trimetrexate, piritrexim, metoprine and pyrimethamine) and thymidylate synthase (AG337, AG377 and AG331). Lipophilic antifolate resistance was determined using growth inhibition assays upon 72 h drug exposure. Cells overexpressing these mutant efflux transporters displayed up to 106-fold resistance to lipophilic antifolates relative to untransfected cells; this resistance was reversed by the specific and potent ABCG2 efflux inhibitor Ko143. In contrast, cells overexpressing the WT R482 ABCG2 exhibited either no or only a low-level of lipophilic antifolate resistance. These results provide the first evidence that overexpression of the mutant G482- and T482 but not the WT R482 ABCG2 confers a high-level of resistance to lipophilic antifolates. The high membrane partitioning of lipophilic antifolates along with the large confinement of ABCG2 to the plasma membrane suggest that these mutant ABCG2 transporters may possibly recognize and extrude lipophilic antifolates from the lipid bilayer. The potential implications to cancer chemotherapy as well as the mechanism of anticancer drug extrusion by these mutant exporters are discussed.

Towards species-specific antifolates.

Dihydrofolate reductase (DHFR) plays an essential role in cellular biochemistry and has been a well-recognized drug target for over fifty years. Antifolate inhibitors of DHFR, including clinically used therapeutics such as methotrexate, trimethoprim, and pyrimethamine have been successful as anticancer, antibacterial, antifungal and antiparasitic agents. As resistant strains of these microorganisms evolve and as new disease threats arise, the need for new antifolates that are potent and specific for infectious organisms becomes more pressing. Several new antifolates have been reported over the past decade; many of these are potent against a particular species of DHFR, but achieving the goal of potency and selectivity has proven to be more difficult. This review will describe recent advances in attaining species selectivity in developing new antifolates. Specifically, advances in developing inhibitors against Pneumocystis jirovecii and Plasmodium falciparum, the causative agents in pneumocystis pneumonia and malaria, respectively, will be presented.

Reduction in brain infarction by augmentation of central histaminergic activity in rats.

Inflammation is a factor in the aggravation of reperfusion injury after cerebral ischemia. Since histamine H(2) receptor stimulation suppresses inflammatory reactions, effects of the central histaminergic activation on brain infarction were examined in rats. Focal cerebral ischemia for 2 h was provoked by transient occlusion of the right middle cerebral artery, and the infarct size was determined by 2,3,5-triphenyltetrazolium chloride stain after 24 h. Effects of postischemic administration of thioperamide, an H(3) antagonist, and metoprine, an inhibitor of histamine-N-methyltransferase, were evaluated in rats treated with l-histidine, a precursor of histamine. Furthermore, effects of these agents on changes in the striatal histamine level were examined by a microdialysis procedure. Focal ischemia provoked marked damage in rats treated with l-histidine (1000 mg/kg) alone. Administration of l-histidine (1000 mg/kg) with either thioperamide (5 mg/kg) or metoprine (10 mg/kg) alleviated brain infarction. The size of brain infarction was 27% and 10% of that in animals treated solely with l-histidine, respectively. The combination treatment with thioperamide and metoprine decreased the size of brain infarction in rats given l-histidine (500 mg/kg), although protective effects were not clear without l-histidine. A marked increase in the histamine concentration was observed in the histidine plus metoprine group, the value being 363% of that in the saline-injected group after 2-3 h. The histamine concentrations in the histidine group and histidine plus thioperamide group were 188% and 248%, respectively. These findings indicate that facilitation of central histaminergic activity reduced the brain infarction.

Structural basis for inhibition of histamine N-methyltransferase by diverse drugs.

In mammals, histamine action is terminated through metabolic inactivation by histamine N-methyltransferase (HNMT) and diamine oxidase. In addition to three well-studied pharmacological functions, smooth muscle contraction, increased vascular permeability, and stimulation of gastric acid secretion, histamine plays important roles in neurotransmission, immunomodulation, and regulation of cell proliferation. The histamine receptor H1 antagonist diphenhydramine, the antimalarial drug amodiaquine, the antifolate drug metoprine, and the anticholinesterase drug tacrine (an early drug for Alzheimer's disease) are surprisingly all potent HNMT inhibitors, having inhibition constants in the range of 10-100nM. We have determined the structural mode of interaction of these four inhibitors with HNMT. Despite their structural diversity, they all occupy the histamine-binding site, thus blocking access to the enzyme's active site. Near the N terminus of HNMT, several aromatic residues (Phe9, Tyr15, and Phe19) adopt different rotamer conformations or become disordered in the enzyme-inhibitor complexes, accommodating the diverse, rigid hydrophobic groups of the inhibitors. The maximized shape complementarity between the protein aromatic side-chains and aromatic ring(s) of the inhibitors are responsible for the tight binding of these varied inhibitors.

Heme-reversible impairment of CYP2B1/2 induction in heme-depleted rat hepatocytes in primary culture: translational control by a hepatic alpha-subunit of the eukaryotic initiation factor kinase?

The role of heme in the phenobarbital-mediated induction of CYP2B1/2 was reexamined in rat hepatocytes in monolayer culture, acutely depleted of heme by treatment with either 3,5-dicarbethoxy-2,6-dimethyl-4-ethyl-1,4-dihydropyridine (DDEP) or N-methylprotoporphyrins (NMPP). The findings revealed that such acute hepatic heme depletion markedly impaired CYP2B1/2 protein induction, an effect that was reversible by heme resupplementation. However, TaqMan analyses of hepatic mRNA isolated from these heme-depleted cells revealed that this impairment was not due to faulty transcriptional activation of either CYP2B1 or CYP2B2 gene expression as previously proposed, thereby confirming literature reports that heme is not a transcriptional regulator of the CYP2B1/2 gene. In contrast, the rate of de novo CYP2B1/2 protein synthesis was found to be dramatically inhibited in both DDEP- and NMPP-treated hepatocytes. Concurrently, a marked (>80%) suppression of de novo hepatocellular protein synthesis was also observed, along with a significantly enhanced phosphorylation of the alpha-subunit of the eukaryotic initiation factor eIF2 (eIF2alpha), as monitored by the phosphorylated eIF2alpha/total eIF2alpha ratio in these heme-depleted cells. Indeed, the parallel reversal of all these three effects by heme supplementation suggests that this impaired CYP2B1 induction most likely stems from blocked translational initiation resulting from the activation of a heme-sensitive eIF2alpha kinase. Such global suppression of hepatic protein synthesis may disrupt a myriad of vital cellular functions, thereby contributing to the clinical symptoms of acute hepatic heme-deficient states such as the hepatic porphyrias.

Stoichiometric selection of tight-binding inhibitors by wild-type and mutant forms of malarial (Plasmodium falciparum) dihydrofolate reductase.

A simple method for screening combinatorial and other libraries of inhibitors of malarial (Plasmodium falciparum) dihydrofolate reductase (PfDHFR) has been developed, based on the affinities of the inhibitors with the enzyme. In the presence of limiting amounts of the enzyme, a number of inhibitors in the library were bound to extents reflecting the relative binding affinities. Following ultrafiltration and guanidine hydrochloride treatment to release bound inhibitors, the amounts of free and bound inhibitors could be determined by high-performance liquid chromatography and liquid chromatography-mass spectrometry. The differences in the patterns reflected the binding of high-affinity components compared with the other members in the library. A good correlation was found between the inhibition constants (Ki values) and the extent of binding of inhibitors to wild-type, double (C59R+S108N) and quadruple mutant (N51I+C59R+S108N+I164L) of PfDHFR, as well as human DHFR. In addition to identifying lead components of the libraries with high affinities (low Ki values) and stabilities (low k(off) rates), this simple method also provides an alternative way for quickly and accurately calculating enzyme binding affinities of inhibitors in combinatorial chemical libraries.

Role of histaminergic neurons in development of epileptic seizures in EL mice.

The EL mouse is an animal model for hereditary temporal lobe epilepsy. When the mice receive weekly vestibular stimulation, e.g., 30 "tosses", 10-15 cm vertically, they start to convulse after 1-2 weeks. The aim of this study was to evaluate the role of the histaminergic neurons in the regulation of seizure development in the EL mice. The obtained results indicated that administration of either histidine, a substrate for histamine synthesis, or metoprine (2,4-diamino-5-(3,4-dichlorophnyl)-6-methyl-pyrimidine), an inhibitor of histamine N-methyltransferase (HNMT), retarded the onset of seizure episodes in the mice. The co-administration of histidine and metoprine caused a more marked delay in it. The histamine levels in the brain significantly increased in response to any of these treatments. The intraperitoneal injection of diphenhydramine, a H1-antagonist accelerated the initiation of seizure episodes in the mice, whereas thioperamide, a H3-antagonist caused a delay in the response. There were significant increases in the brain histamine levels upon injection of any of these drugs with concomitant rises in the activity of the histidine decarboxylase (HDC). These results, taken together, suggest that the histaminergic neurons play crucial roles in the development of seizures in the EL mice. They inhibit convulsion in a H1-dependent fashion, while the neurons enhance it in a H3-receptor-mediated way.